Electrophoresis refers to the motion of charged objects through a fluid under the influence of an electric field. This phenomenon can be used to separate objects according to their electric and hydrodynamic properties, and a number of techniques for exploiting this are in widespread use. The objects to be separated, commonly proteins or other biomolecules, are typically suspended in a fluid such as a buffer solution or a gel. A small section of the solution containing the objects is placed at the beginning of a separation channel containing a fluid or gel and subsequently a constant electric field is generated along the channel. Under the influence of this field the said objects start moving towards the opposite end of the channel. As they migrate through the fluid, they experience differential hydrodynamic force depending on their shape and size. Due to this different hydrodynamic force applied to them, the objects move with different terminal velocity depending on their individual characteristics and thus they separate and form “bands”. Due to their different terminal velocities, the distance between the bands increases with time.
A band is essentially a group of objects having like electrical and hydrodynamic properties. One of the key disadvantages of electrophoresis is the fact that the bands, as they move at their terminal velocity, undergo thermal diffusion. This makes the bands broaden with time reducing the resolution of the separation.
Various known implementations of electrophoresis are reviewed in “Cyclic electrophoretic and chromatographic separation methods”, Eijkel et al, Electrophoresis 2004, 25, 243-252.
There are also a number of variants to the conventional electrophoresis method that attempt to limit the thermal diffusion.
One such method is Iso-Electric-Focussing (IEF) which deploys a pH gradient in the separation channel. As the objects move through the fluid under the influence of a constant electric field, their apparent charge changes due to the changing pH along the channel. Each object, depending on its charge characteristics, moves until a point where its apparent charge is zero. This is called the “isoelectric point”. At that point the object stops moving as it reaches an equilibrium position. Each object with different charge characteristics stops at a different point along the channel and thus a separation of the objects takes place. The bands of objects can then be detected and investigated, for example by imaging.
The advantage of this method is the elimination of thermal diffusion and the disadvantage is the limited accuracy of the pH gradient. Another disadvantage is the increased separation times (typically many hours).
Another electrophoresis variant is disclosed in US-A-2002/0043462. Particles are separated by applying a first force resulting from buffer flow through the chamber which is opposed by an electric field gradient. The shape of the static electric field is such that the particles separate into bands along the chamber. The bands represent equilibrium positions at which the net force on each molecule is zero. Once the bands are formed, the applied electric field may be modified so as to manipulate the bands, for example moving a band of interest to an exit point. However as in the case of other known systems, the device relies on a constant flow of buffer through the chamber in order to impose the appropriate hydrodynamic force on each particle and thus achieve successful separation. This leads to a number of problems.
Firstly, the pumping and monitoring equipment required to achieve the highly accurate liquid flow through the channel leads to a potentially expensive and complicated infrastructure. As a result, the systems are typically costly and can be unreliable, comprising a number of complex mechanical components. The accuracy of the buffer flow is essential to the accuracy and resolution of the device.
Secondly, a common problem associated with the use of flowing liquids, and experienced heavily in high performance liquid chromatography (HPLC) processes using pressure driven flow, is that the liquid interacts with the channel walls. As a result, the liquid does not flow with a constant velocity across the channel cross-section but rather moves more slowly adjacent to the walls and faster towards the middle of the channel. This creates a parabolic velocity front, which directly affects the band shape of the separated molecules. The greater the departure from a constant velocity front, the wider the bands become, thus reducing resolution in the device. The separation process can be sped up by increasing the buffer flow rate. This is because the time taken for molecules to reach their equilibrium position shortens and hence the separation should complete more quickly. In a first approach, increasing the flow rate should also narrow the stationary bands because the exerted hydrodynamic and electric forces are larger. This would result in improved resolution.
However, as the flow rate is increased, the parabolic flow profile becomes accentuated, tending to cancel out the expected improvement in resolution.
Conventional electrophoresis often involves the use of gels as the separation fluid. The relatively high viscosity reduces diffusion and so improves resolution. However, it is difficult, if not impossible, to use gels with techniques requiring buffer flow (such as the two aforementioned methods) because the gels do not generally flow easily.
What is needed is a technique that effectively controls thermal diffusion and which eliminates the need for a constant flow of separation fluid through the apparatus. Such a method should achieve fast separation and high resolution. The present invention is provided to solve the problems discussed above and other problems, and to provide advantages and aspects not provided by prior electrophoresis methods and systems. A full discussion of the features and advantages of the present invention is deferred to the following summary, detailed description, and accompanying drawings.